Polymeric Nonviral Gene Delivery Systems for Cancer Immunotherapy

Background: Dendritic cell (DC) vaccines have significant potential in cancer immunotherapy. While autologous DCs can be derived from bone marrow, umbilical cord, and peripheral blood, monocyte-derived DC vaccines are most widely tested in clinical trials. However, producing autologous DC vaccines is labor intensive, has large variations among donors, and may not be feasible for patients with impaired cell function or requiring multiple vaccinations. Human pluripotent stem cells (hPSCs) have unlimited expansion potential while maintaining their pluripotency. They are being tested as a novel cell source to derive DCs for clinical application. Lung cancer is the leading cause for all cancer-related mortality. Efficient treatment of lung cancer by DC vaccines could offer great benefits in cancer immunotherapy. This review uses lung cancer as a case study to discuss the application of DC vaccines. Results: DC derivation from hPSCs has been demonstrated with high purity and comparable in vitro functions to autologous DCs derived from monocytes. The differentiation can be achieved either by co-culturing hPSCs with OP9 stromal cells or by the formation of three-dimensional embryoid bodies. As the scalable culture system is critical for hPSC-derived DC production, progress in the scalable culture systems for other hPSC-derived cells is reviewed and the use of relevant systems for hPSC-derived DCs is proposed. Conclusions: hPSCs provide a new source for DC production and have significant implication in DC-based cancer immunotherapy. Their use in clinical trials requires refinement of the culture expansion and differentiation protocols. Development of scalable culture systems is crucial in truly harnessing the potential of the hPSC-based DC immunotherapy in cancer treatment.

Polymeric gene delivery vectors show great potential for the construction of the ideal gene delivery system. These systems harness their ability to incorporate versatile functional traits to overcome most impediments encountered in gene delivery: from the initial complexation to their target-specific release of the therapeutic nucleic acids at the cytosol. Among the numerous multifunctional polymers that have been designed and evaluated as gene delivery vectors, polymers with redox-sensitive (or bioreducible) functional domains have gained great attention in terms of their structural and functional traits. The redox environment plays a pivotal role in sustaining cellular homeostasis and natural redox potential gradients exist between extra- and intracellular space and between the exterior and interior of subcellular organelles. In some cases, researchers have designed the polymeric delivery vectors to exploit these gradients. For example, researchers have taken advantage of the high redox potential gradient between oxidizing extracellular space and the reducing environment of cytosolic compartments by integrating disulfide bonds into the polymer structure. Such polymers retain their cargo in the extracellular space but selectively release the therapeutic nucleic acids in the reducing space within the cytosol. Furthermore, bioreducible polymers form stable complex with nucleic acids, and researchers can fabricate these structures to impart several important features such as site-, timing-, and duration period-specific gene expression. Additionally, the introduction of disulfide bonds within these polymers promotes their biodegradability and limits their cytotoxicity. Many approaches have demonstrated the versatility of bioreducible gene delivery, but the underlying biological rationale of these systems remains poorly understood. The process of disulfide reduction depends on multiple variables in the cellular redox environment. Therefore, the quest to unravel various issues such as the site and time of disulfide bond reduction during the cellular uptake and trafficking have stimulated a number of interesting studies which have employed disulfide compounds with a variety of reducible linkers. Such studies help researchers understand not only how modifications made to disulfides can alter their thiol-disulfide exchange characteristics but also to decipher the effect of the induced changes on the dynamics of the redox environment. This Account discusses current research trends and recent progress in the disulfide chemistry enabling novel and versatile designs of reducible polymeric gene delivery systems. We present strategies for the introduction of disulfide bonds into polymers. These representative examples and their respective outcomes elaborate the benefit and efficiency of disulfides at the individual stages of gene delivery.

It is becoming clear that ionizing radiation positively influences certain immune parameters, which opens the possibility for combining radio- and immunotherapies in cancer treatment. The presence of functionally competent dendritic cells (DCs) is crucial in mounting a successful antitumor immune response. While it has been shown that DCs are relatively radioresistant, few and contradictory data are available on how ionizing radiation alters the functional integrity of these cells. Therefore, our objective was to investigate the effect of whole-body irradiation on the function of splenic DCs. C57Bl/6 mice were irradiated with 0.1, 0.25, and 2 Gy X-rays and changes in the phenotype of splenic DCs were compared to unirradiated controls. An increase was seen in DC surface markers influencing DC-T cell interactions. In vivo cytokine production was determined by direct intracellular cytokine staining. Irradiation with 2 Gy induced a 1.6-fold increase in IL-1α production, while the combination of irradiation and lipopolysaccharide (LPS) treatment induced a 3.9-fold increase, indicating a strong synergism between irradiation and LPS stimulation. Interaction of DCs with effector and regulatory T cells was investigated in a mixed lymphocyte reaction. While DCs from control animals induced stronger proliferation of regulatory T cells, DCs from animals irradiated with 2 Gy induced stronger proliferation of effector T cells. Antigen uptake and presentation was investigated by measuring the capacity of DCs to internalize and present ovalbumine (OVA)-derived peptides on their major histocompatibility complex (MHCI) molecules. Irradiation with 2 Gy did not influence antigen uptake or presentation, while low doses stimulated antigen uptake and reduced the level of antigen presentation. In conclusion, high-dose in vivo irradiation induced increased expression of T cell costimulatory markers, enhanced production of proinflammatory cytokines and a stronger stimulation of effector T cell proliferation than that of regulatory T cells. However, it did not influence DC antigen uptake or presentation. On the other hand, low-dose irradiation increased antigen uptake and lowered antigen presentation of DCs, indicating that low- and high-dose irradiation act on different pathways in DCs.

The immune system and cancer cells interact intricately during the growth of tumors, and the dynamic interplay between immune activation and suppression greatly influences the cancer outcome. Natural killer cells (NK), cytotoxic T lymphocytes (CTLs) and Dendritic cells (DC), employ diverse mechanisms, to combat cancer. However, the challenges posed by factors such as chronic inflammation and the immunosuppressive tumor microenvironment (TME) often hinder immune cells' ability to detect and eliminate tumors accurately. Immunotherapy offers a promising approach, reprogramming the immune system to target and eliminating cancer cells while minimizing side effects, enhancing immune memory, and lowering the risk of metastasis and relapse compared to traditional treatments like radiation and surgery. Nanotechnology presents a potential solution by enabling safer, more efficient drug delivery through nanoparticles. These nanoengineered drugs can be tailored for controlled activation and release. Improving TME characters holds potential for enhancing personalized immunotherapy and addressing T cell availability issues within tumor sites, particularly when combined with existing therapies. This review discusses TMEs and the strategies to overcome immunosuppression in TME, and various immune cell-based strategies to improve antitumor response. It also focuses on the strategies for constructing microenvironment responsive nanoplatforms based upon the factors present at higher levels in TME like acidic pH, hypoxia facilitated by poor oxygen supply, higher expression of certain enzymes, and other factors such light, ultrasound and magnetic field. Combination immune therapies combined with immunotherapy include photodynamic therapy, photothermal therapy, chemotherapy, gene therapy and radiotherapy, revealing a high level of anticancer activity in comparison to a single therapy, enhancing immunogenicity, promoting therapeutic efficacy, and lowering metastasis. In conclusion, cancer immunotherapy is a potential technique to combat cancer cells and boost the immune system, hindering their growth and recurrence. In order to prevent cancer, it helps the immune system target cancer cells selectively and strengthens its long-term memory. Clinical trials are extending the application of immunotherapy and identifying strategies to improve the immune system tumor-fighting capabilities. Immunotherapy has enormous promise and gives hope for more successful cancer treatment.

Dendritic cell-based immunotherapy for colon cancer using an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope from tumor-associated antigen 90K

Non-viral vectors have been widely proposed as safer alternatives to viral vectors, and cationic polymers have gained increasing attention because they can form self-assembly with DNA. Chitosan is also considered to be a good candidate for gene delivery systems, since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic potential. However, low solubility and transfection efficiency need to be overcome prior to clinical trial. In this work, we focus on alkyl modified chitosan which might be useful in DNA condensing and efficient gene delivery. N, N- Diethyl N- Methyl (DEMC) and N- Triethyl Chitosan (TEC) were synthesized from chitosan polymer. In order to optimize the polymers for gene delivery, we used FITC-dextran (FD). Then the optimized polymer concentrations were used for gene delivery. Fluorescent microscope was used, in order to evaluate the polymers' efficiency for gene delivery to human embryonic kidney epithelial cells (HEK 293T). This modification increased chitosan's positive charge, thus these chitosan derivatives spontaneously formed complexes with FD, green fluorescence protein plasmid DNA (pEGFP), red fluorescence protein plasmid DNA (pJred) and fluorescent labeled miRNA .RESULTS gained from fluorescent microscope showed that TEC and DEMC were able to transfer FD, DNA and miRNA (micro RNA) to HEK cell line. We conclude that these chitosan derivatives present suitable characteristics to be used as non-viral gene delivery vectors to epithelial cells.

Research Highlights: Highlights From The Latest Articles in Nanomedicine

With the huge therapeutic potential, cancer immunotherapy is expected to become the mainstream of cancer treatment. In the current field of cancer immunotherapy, there are mainly five types. Immune checkpoint blockade therapy is one of the most promising directions. Adoptive cell therapy is an important component of cancer immunotherapy. The therapy with the cancer vaccine is promising cancer immunotherapy capable of cancer prevention. Cytokine therapy is one of the pillars of cancer immunotherapy. Oncolytic immunotherapy is a promising novel component of cancer immunotherapy, which with significantly lower incidence of serious adverse reactions. The recent positive results of many clinical trials with cancer immunotherapy may herald good clinical prospects. But there are still many challenges in the broad implementation of immunotherapy. Such as the immunotherapy cannot act on all tumors, and it has serious adverse effects including but not limited to nonspecific and autoimmunity inflammation. Here, we center on recent progress made within the last 5 years in cancer immunotherapy. And we discuss the theoretical background, as well as the opportunities and challenges of cancer immunotherapy.

256. Importance of Temporal Control of HSVtk Gene Therapy in Combination with T Cell Based Therapies for Cancer

Importance of the field: Due to the absence of early diagnosis, the highly invasive and metastatic features and the lack of effective therapeutic modalities, the prognosis of patients with pancreatic cancer is poor. Gene therapy is currently regarded as a potential and promising therapeutic modality for pancreatic cancer.Areas covered in this review: This article summarizes an update of gene therapy approaches and reviews the latest progress in gene delivery systems that have been tested on pancreatic cancer.What the reader will gain: The treatment effectiveness of gene combination therapy is better than that of the regulation of single-gene or single gene therapy approaches. Naked DNA is limited because of degradation by intracellular and extracellular nucleases. Virus vectors show high transfection efficiency but are limited due to immunogenicity, inflammatory response and potential carcinogenicity. Non-viral vectors, such as cationic polymers or inorganic nanoparticles, show an important feature that they can be easily modified, and the progress of materials science will provide more and better non-viral vectors, accordingly improving the efficiency and safety of gene therapy, which will make them the most promising vectors for pancreatic cancer.

Eighth International Nanomedicine and Drug Delivery Symposium (NanoDDS'10)

ROS-responsive fluorinated polycations as non-viral gene vectors

Sex differences in cancer susceptibility and mortality are well documented. In general, men have a higher cancer incidence and poorer outcomes when compared to women. Of immunologic relevance, and in contrast to cancer, women account for almost 80% of the autoimmune cases reported. These findings suggest male sex is a ‘risk factor’ for cancer but protective against autoimmunity and also highlight the potential involvement of sex hormones in regulating the immune response. Several recent publications have put a spotlight on androgen-mediated immune regulation and its impact on anti-tumor immunity. Guan and colleagues demonstrated that blockade of sex hormones in prostate tumor-bearing mice resulted in improved adaptive immune responses via direct modulation of CD8+ T cell-intrinsic androgen receptor signaling. Androgen-deprivation led to increased interferon gamma (IFNγ) expression by T cells and improved overall anti-tumor immunity. Additionally, others have shown using non-reproductive cancer mouse models (melanoma, bladder, colon and liver) that androgen receptor signaling promotes T cell exhaustion, and inhibits the activity and stemness of anti-tumor CD8+ T cells. Importantly, there is a clear role for androgen-mediated immune regulation in non-reproductive human cancer given a recent publication showing improved progression-free survival and overall survival in female versus male melanoma patients who were treated with neoadjuvant BRAF/MEK-targeted therapy. Follow-up preclinical mouse models showed significantly higher expression of the androgen receptor in tumors of both male and female BRAF/MEK-treated mice versus control and pharmacological inhibition of androgen receptor signaling improved responses to BRAF/MEK-targeted therapy in both male and female mice. Taken together, these publications stress the importance of understanding androgen-mediated immune control in both male and female cancer patients and the broad applicability of this signaling pathway in tissues including, but also beyond, the reproductive tract. There is mounting evidence to support a role androgen signaling in T cell biology. However, professional antigen presenting cells such as dendritic cells (DCs) are an essential part of generating robust anti-tumor T cell immunity were not addressed in these studies. Notably, DCs have been reported to be sensitive to androgens. Castration studies in mice found that removal of testosterone increased antigen presentation and expression of co-stimulatory molecules on DCs. In addition, hypogonadal men are at elevated risk of developing autoimmunity. Isolation of DCs from these patients showed a significant increase in DC maturation. HIV infected DCs derived from males produced significantly less type I interferon compared to female patients, resulting in weaker CD8 T cell responses against the virus. These data establish that androgens can shape DC function, yet they fall short in identifying the mechanisms of how androgens regulate DCs and the impact this may have on anti-tumor immunity. In our initial interrogation of a single-cell RNA sequencing dataset of human prostate cancer patients, we found that human tumors with low androgen receptor activity had increased IFNγ pathway activity and that IFNγ activity was positively associated with an increase in antigen presentation. Importantly, IFNγ is a cytokine that strongly induces antigen processing and presentation by antigen presenting cells and androgen deprivation therapy increases T cell derived IFNγ in tumor bearing mice suggesting that androgen receptor activity may also regulate antigen presentation. Furthermore, our analysis showed that elevated androgen receptor activity correlated with lower antigen presentation, conceivably through suppression of DC function. We hypothesized that inhibition of androgen receptor-mediated suppression of DC function would improve antigen presentation and anti-tumor T cell responses. Here we demonstrate using a B16 melanoma model that male and female DCs, both bone marrow-derived and those sorted from murine steady-state skin and melanoma tumors, express androgen receptor. Interestingly, there is a striking upregulation of androgen receptor expression specifically on type 1 conventional DCs (cDC1s) following uptake of tumor-derived antigen in vivo. cDC1s are a subset of DC that is particularly specialized in cross-presentation of exogenous antigens on MHCI. In this way, cDC1s are dominant drivers of CD8+ T cell responses and, via this function, have been shown to be essential for anti-tumor immunity. Further by inhibiting androgen receptor signaling in vitro using the clinically relevant inhibitor, enzalutamide, we find that bone marrow-derived DCs have increased phagocytic activity as shown by fluorescent bead uptake. Next, we injected male mice with B16 melanoma tumors and, after allowing the tumors to establish, we treated them with degarelix to reduce testosterone levels and enzalutamide to further inhibit androgen receptor signaling. On day 15 we sacrificed the mice, measured the tumors and interrogated DC function by flow cytometry. Strikingly, we found that inhibition of androgen receptor signaling in vivo significantly increased tumor-derived antigen uptake by cDC1s and, importantly, this correlated with improved tumor control as seen by significantly smaller tumors upon androgen receptor inhibition. These data provide insight into the regulation of DCs within the tumor microenvironment and how suppressing inhibitory hormone molecules may lead to better antigen presentation and more effective downstream T cell responses. Cancer immunotherapy is designed to generate an autoimmune-like response against an individual’s cancer. Given the striking sexual dimorphism in both autoimmunity and cancer, there is a clear and pressing need to investigate the impact of sex hormones on the effectiveness and function of cancer immunotherapy. And while myeloid cell targeted therapies and DC-based anti-tumor vaccines hold great promise for boosting anti-tumor T cells responses, current clinical use and efficacy has been limited pointing to an urgent need for improved vaccine and therapeutic design. This will rely on a better understanding of DC activation in this context. Our data indicates that there is a role for androgen receptor signaling in DC function and highlights the potential impact of DC-intrinsic androgen receptor inhibition on optimal DC vaccine design and the generation of more productive DC targeted immunotherapies. Citation Format: Kasidy Brown, Fanny Polesso, Victoria Schuster, Amy Moran, Megan Ruhland. Dendritic cell intrinsic androgen receptor signaling reduces dendritic cell function and anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG01.

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DCs are used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferons (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to URC alone, or in combination with TNF-α, IFN-α and IFN-γ. All three cytokines induced phenotypically mature DC. The expression levels of CD38 and CCR7 on URC-primed DC were enhanced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. DC matured with URC plus IFN-γ produce high levels of IL-12p70 upon CD40 triggering. IFN-γ secretion from naïve T cells co-cultured with URC-primed DC also augmented by IFN-γ. In addition, DC maturated with URC plus IFN-γ has an intermediate migratory capacity towards CCL19 and CCL21. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α and IFN-α. These data support that DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3828.